This is a renewal application to continue studies on the molecular regulation of apoprotein B (apoB) degradation. ApoB is the predominant protein component of the atherogenic lipoproteins. Thus, knowledge of the regulation of the assembly and secretion of apoB-lipoproteins is not only of fundamental interest, but is also medically relevant. An important control of the net secretion of apoB from cells of hepatic origin is the level of its pre-secretory degradation, one of the many unusual features of the biosynthesis and assembly of apoB into lipoprotein particles. We have recently established powerful cell-free systems to complement studies in cultured cells and animal models to identify and investigate the molecular factors that target apoB to degradation by proteasomal and non-proteasomal mechanisms as well as those that mediate apoB exit from the ER as part of a lipoprotein particle. The application is divided into 3 proposed aims: Aim 1 is to determine the spectrum of factors regulating the proteasome-mediated ER-associated degradation (ERAD) of apoB. We have previously established that cytosolic Hsp70 and Hsp90 promote ERAD of apoB and propose that known Hsp70 and Hsp90 co-chaperones and accessory factors also play important roles. Aim 2 is to determine the role of oxidant stress in the post-ER, non-proteasomal degradation of apoB induced by n-3 fatty acids. N-3 fatty acids are known hypolipidemic agents and we have recent data that in hepatic cells they stimulate a post-ER, non-proteasomal, degradative pathway (with remarkable similarity to the one induced by insulin) that is blocked by anti-oxidants. The molecular characteristics of n-3-stimulated apoB degradation, particularly with regard to oxidant stress, and its relationship to the insulin-stimulated process, will be tested in cell culture systems and in 2 recently described mouse models of human diseases, one of familial combined hyperlipidemia (and which has increased hepatic antioxidant levels) and one of insulin resistance (the Akt2 KO mouse). Aim 3 is to determine the factors regulating the ER-exit of apoB-containing lipoproteins. Based on classical studies of protein trafficking, the large size and other properties of apoB lipoproteins make it likely that there are novel features that drive their packaging and exit from the ER. Using cell-free model systems (established, to our knowledge, for the first time for this purpose), and hepatic cells, we will identify the molecular characteristics of the ER-exit process.